Purification and solubilization of paired helical filaments from Alzheimer brains.
The purpose of the present study was to develop a purification and solubilization method, compatible with current amino acid sequencing techniques, for paired helical filaments (PHFs) derived from patients with Alzheimer's disease. We have developed a mild procedure that subjects conventionally isolated PHFs to Tris/borate/sodium dodecyl sulfate/2-mercaptoethanol electrophoresis and results in the separation of the relatively insoluble PHF structures from both copurifying contaminating proteins and solubilized PHF-associated proteins. At the end of 4.5 h of electrophoresis, the purified insoluble fraction had an amino acid composition that was invariant during subsequent electrophoresis. Electron microscopy revealed an intact PHF structure before and after electrophoresis but no evidence of any other structures in the insoluble fraction, a result consistent with the removal of PHF-associated proteins from the filament structure. Isolated insoluble filament structures displayed an enhanced immunoreactivity with antibodies raised against purified PHFs in other laboratories, when compared with the fraction not subjected to electrophoresis in enzyme-linked immunosorbent assays. Solubilization of the relatively insoluble PHFs was accomplished by extending the time of electrophoresis beyond the 4.5 h required for purification. Additional electrophoresis for 34.5 h solubilized 88% of the purified, relatively insoluble PHFs. This resulted in the identification of four major protein bands between Mr values of approximately 50,000 and 70,000 on sodium dodecyl sulfate-polyacrylamide electrophoresis gel analysis, with a predominant band with an Mr of approximately 66,000. A slow fragmentation of the PHF ultrastructure occurred during this time, as judged by electron microscopy. This purification technique will permit the isolation of consistently reproducible protein fragments from solubilized PHFs, which may be used for subsequent sequence analysis.
Vogelsang
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GD
Alzheimer's Research Center, University of Cincinnati College of Medicine, Ohio 45267-0524.
Zemlan
F P
FP
Dean
G E
GE
D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology*; D000596:Amino Acids / Q000032:analysis; D001921:Brain / Q000648:ultrastructure*; D002458:Cell Fractionation / Q000379:methods; D002540:Cerebral Cortex / Q000648:ultrastructure; D003599:Cytoskeleton / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007382:Intermediate Filaments / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000302:isolation & purification; D013702:Temporal Lobe / Q000648:ultrastructure
D000596:Amino Acids; D009419:Nerve Tissue Proteins